Manipulating Protein Stability with Small Molecules: Applications in studying biological systems and accessing new drug targets

The development of new drugs has traditionally focused on the discovery and optimization of small molecule inhibitors. Despite the success of small molecule inhibitors in treating many diseases, it has been estimated that as much as 80% of all proteins encoded by the human genome cannot be targeted by this therapeutic modality (i.e. are undruggable). The vast size of the undruggable proteome results in a need for a general method to develop small molecules that can alter the activity of any target protein, not just those with enzymatic activity and tractable active sites. Towards that end, we developed hydrophobic t&barbelow;ags (HyTs), a class of small molecules that bind to a protein of interest, thermodynamically destabilize the target protein, and induce the protein's degradation by the ubiquitin-proteasome system.;Chapter 1 describes our work developing a HaloTag-based small molecule microarray (SMM) technique that exhibits increased sensitivity in detecting protein-small molecule binding interactions compared to previous SMM techniques described in the literature. We provide a head-to-head comparison between HaloTag-based SMMs and the traditional antibody-based SMMs, and we introduce the possibility of multiplex screening. A multiplex screen for small molecule ligands specific to the protein tyrosine phosphatase 1B trapped in its second transition state yielded ligands that interact with orthovanadate-bound PTP1B. SMM screening should facilitate the discovery of small molecule ligands that bind to any protein of interest. These small molecules may then be derivatized into hydrophobic tags that induce the degradation of the proteins of interest in cells.;Chapter 2 describes our work characterizing hydrophobic tags (HyTs) and protein stabilizers using the model protein, HaloTag2. Our work studying this system yielded insights into the mechanisms through which HyTs and protein stabilizers can exert their effects in cells. HyT36 binds to the HaloTag2 protein and induces its thermodynamic destabilization, leading to its association with Hsp70 and subsequent degradation via the ubiquitin-proteasome system. Conversely, the HaloT&barbelow;ag S&barbelow;tabilizer (HALTS1) binds to the HaloTag2 protein in the same active site as HyT36 but stabilizes the protein and increases its levels in the cell. We also discuss how the combination of HyT36 and HALTS1 led us to a HyT/HALTS system whereby any protein of interest can be fused to HaloTag2 and have its cellular levels increased 4-fold or decreased 10-fold using the corresponding small molecule.;Chapter 3 describes our initial work exploring whether hydrophobic tags can be targeted to non-cytosolic proteins. For this initial work, we localized the HaloTag2 protein to the endoplasmic reticulum. Destabilizing this Endoplasmic Reticulum Localized HaloTag2 (ERHT) protein using HyT36 did not induce significant degradation but did activate the unfolded protein response (UPR). The ERHT system provided a unique opportunity for us to study activation of the UPR without inducing apoptosis. Treatment of ERHT-expressing cells with HyT36 induced an acute, resolvable endoplasmic reticulum stress that resulted in transient UPR activation without induction of apoptosis. Transcriptome analysis of late-stage responses to this UPR stimulus revealed a link between UPR activity and estrogen signaling: UPR signaling induces estrogen receptor transcriptional activity, which modulates future sensitivity to endoplasmic reticulum stress. Estrogen receptor activation desensitizes MCF7 cells to endoplasmic reticulum stress, and conversely, estrogen receptor inhibition sensitizes MCF7 cells to endoplasmic reticulum stress. Additionally, estrogen receptor inhibition in multiple myeloma cell lines sensitized those cells to UPR activation and apoptosis induced by the proteasome inhibitor, epoxomicin.;Chapter 4 takes a detour from the development of hydrophobic tags to instead focus on the development of a novel, bio-orthogonal protein-small molecule ligand pair. Using crystal structures of HALTS1 bound to HaloTag7, we developed a new small molecule (HALTS2) that binds specifically to a mutated HaloTag7 protein containing W151L and N51L mutations. This mutated protein is incapable of binding to chloroalkanes, meaning that the interaction between HALTS2 and the mutant protein is completely orthogonal to the interaction between chloroalkane-containing compounds and HaloTag7. These two protein-ligand pairs can be used together in future work focused on chemical-induced dimerization.;Finally, chapter 5 offers my perspective on the future of HyTs and protein stabilizers as therapeutic modalities. I discuss the most critical remaining questions that should be addressed in the future development of HyTs. it is our hope that the pursuit of hydrophobic tags and protein stabilizers will eventually enable the development of new drugs to target currently untreatable diseases.